The present invention relates to the in vivo effects of profilin in plant cells. In particular, it has been found that manipulation of the level of expression of profilins in plants can lead to useful alterations in plant phenotype.
Actin is the major cytoskeletal protein of most eukaryotic cells and actin filaments are often the primary determinants of cell shape and movement. The polymerization and depolymerization of actin filaments inside the non muscle cells are highly regulated, both spatially and temporally, to give the cell the ability to rearrange its cytoskeleton drastically within minutes in response to external stimuli or at a point of the cell cycle. In addition to the roles in cell shape and movements, the actin cytoskeleton in plants has been shown to play a role in cytoplasmic streaming, cytokinesis, cell expansion and plant development (Williamson, 1993; Meagher and Williamson, 1994). To achieve these dynamic rearrangements the cell relies on a variety of actin-binding proteins.
Profilin is an ubiquitous actin-monomer binding protein whose homologs are present in organisms ranging from fungi and amoebae through higher plants and mammals. They are low molecular mass (12-15 Kd), actin-binding proteins involved in the regulation of actin dynamics. Apart from actin-binding, profilins also bind to phosphatidylinositol 4,5-bisphosphate (PIP2)(Sohn et al., 1995), poly-L-proline (Bjorkegren et al., 1993) and a proline rich VASP (Reinhard et al., 1995; Haffner et al., 1995). Evidence suggests that profilin is a multi-functional protein (Haarer et al., 1990) which exerts both positive and negative effects on the actin polymerization (Theriot and Mitchison, 1993) and is involved in signal transduction pathways (Sohn and Goldschmidt-Clermont, 1994).
In vitro studies showed that profilin can lower the critical concentration for actin polymerization, stimulate polymerization and promote nucleotide exchange on actin monomers (for reviews see Theriot and Mitchison, 1993; Sohn and Goldschmidt-Clermont, 1994 and Sun et al., 1995). In vivo studies on profilin functions also yielded mixed results. Profilin mutations affected multiple actin-dependent processes in Drososphila (Verheyen and Cooley, 1994), blocked yeast cell budding (Haarer, 1990) and cytokinesis, as they cannot assemble the contractile rings, in Schizosaccharomyces pombe (Balasubramanian et al., 1994). Recent studies on Dictyostelium discoideum showed 60 to 70% increase in F-actin content at the rim below the plasma membrane when both profilins are deleted (Haugwitz et al., 1994). Microinjection of profilin into animal (Cao et al., 1992) or plant (Staiger et al., 1994) cells depolymerized actin. Profilin overexpression in stably transformed cells increased polymerization of actin at the cell periphery by prolonging cortical actin filament half-life (Finkel et al., 1994).
In addition to the above functions, profilin has been shown to be involved in at least two signal transduction pathways. They are the growth factor mediated receptor tyrosine kinase pathway and the Ras/G protein signaling pathway (for a review see Sohn and Goldschmidt-Clermont, 1994). Profilin has shown to bind PIP2 with high affinity and inhibits unphosphorylated phospholipase C (Goldschmidt-Clermont et al., 1990 and Drobak et al., 1994). This inhibition is overcome by growth factor mediated phosphorylation of phospholipase C (Goldschmidt-Clermont et al. 1991). The cyclase associated protein (CAP)/Srv2p is a component of the Ras/G protein signaling pathway and an yeast mutant of CAP was rescued by profilin, which suggested a possible role for profilin in this pathway (Vojtek et al., 1991). Profilin can bind to actin or PIP2 simultaneously with poly-L-proline proteins and proline rich sequences found in many proteins (Reinhard et al., 1995). This interaction with proteins that have proline rich sequences suggested a role for profilin in mediating membrane-cytoskeletal communications by physically attaching signaling proteins to plasma membrane and/or cytoskeleton.
In plants profilin has been identified and characterized in several species including maize pollen and tobacco (Staiger et al., 1993 and Mittermann et al., 1995), leaves and root nodules of Phaseolus vulgaris (Vidali et al., 1995) and Arabidopsis thaliana (Huang et al., 1996 and Christensen et al., 1996). Plant profilins have been shown to bind plant and animal actin In vitro (Valenta et al., 1993; Giehl et al., 1994 and Ruhlandt et al., 1995). Recently Perelroizen et al. (1996) reported that Arabidopsis profilin, like other profilins, can bind G-actin and promote assembly of actin filaments at the barbed end in vitro. In contrast to other profilins, Arabidopsis profilins do not accelerate the nucleotide exchange on actin. Despite these advances in plant profilin studies, there have been no reports so far on the in vivo functional analysis of profilins in plants.
We have found that over-production of profilin in plants or plant cells, preferably achieved by transformation of a plant or plant cell with a chimeric profilin gene that is highly expressed in plants, can cause increased cell elongation in growing plants. One of the results of this effect is a plant with a xe2x80x9ctallxe2x80x9d phenotype compared to the non-transgenic control plant. Another result is elongation of the root and root hair system of the plant.
We have also found that under-production of profilin, preferably achieved by reducing expression of native profilin in a plant (such as by the use of antisense RNA), can cause decreased cell elongation. One of the results of this is a xe2x80x9cdwarfxe2x80x9d phenotype.
It is also possible to direct over-expression or under-expression to specific tissues of the plant, such as root tissues. This results in a selective effect on the growth of that tissue. In one embodiment, a profilin coding sequence is linked to a root-specific promoter, resulting in elongation of roots, and particularly root-hairs. This increase in root surface area increases the plant""s capacity to absorb water and nutrients, making it more drought-resistant, for instance, or less dependent on chemical fertilizers.
Control of the level of profilin production can also effect control of plant flowering time. It has been found that over-production of profilin by a plant leads to a delay in flowering time, while under-production of profilin promotes early flowering.
Such control of plant stature and flowering time has many applications in the creation of agricultural and horticultural varieties of plants.
The present invention takes advantage of the in vivo functions of plant profilins to effect changes in plant morphology, growth and flowering. It has been found that altering the level of expression of profilin in a plant cell leads to alterations in cell morphology and in cell elongation. At an organismic level, these changes can effect the growth habit of the plant, as well as effect the onset of flowering.
In an embodiment of the invention, a study was made of the in vivo functions of the Arabidopsis thaliana profilin-1 gene. Transgenic plants over- and under-expressing profilin-1 were generated by transforming wild type Arabidopsis plants with sense or antisense constructs under the control of a 35S promoter. Etiolated transgenic seedlings under-expressing profilin-1 exhibited overall stunted growth, short hypocotyl and at low temperature they were only 15 to 20% long when compared with the wild type plants. Light grown plants also showed the reduced growth and these plants flowered early. Light and electron microscopic observations of the hypocotyl surface and sections of the etiolated seedlings revealed that the surface of these seedlings were ruffled with some electron dense particle accumulation and enlarged epidermal, cortical and endodermal cells. The epidermal and cortical cells also showed defective vesicular transport with fibrous and electron dense particles and vesicles accumulating in the cytoplasm and close to the plasma membrane. On the other hand, the etiolated transgenic seedlings over-expressing profilin-1 were slightly bigger than the wild type in light and at normal and low temperature. Microscopic analysis of these seedlings showed no obvious changes compared to the wild type, except that the epidermal, cortical and endodermal cells were bigger. Light grown plants showed a flowering time delayed by at least by a week. Actin staining on the etiolated seedlings over or under expressing profilin-1 showed normal actin staining pattern with networks in the cotyledon cells and petiole cells. Transgenic seedlings harboring the profilin-1 promoter-Gus construct showed the Gus expression as a ring at the elongating zone of the root meristems and the root hairs. These changes were inheritable, even to the fourth generation.
It is frequently desirable in horticulture and agriculture to have plant varieties that have a taller or shorter growth habit in respect to the xe2x80x9cwild typexe2x80x9d of the species or variety. For instance, in rice a xe2x80x9ctallxe2x80x9d variety is better able to survive flooding conditions by growing higher above the water level. Conversely, a xe2x80x9cdwarfxe2x80x9d variety of beans grows as a bush instead of a vine, taking up less space, requiring little or no physical support during growth and facilitating harvest. Tall and dwarf ornamental varieties are also much sought after. Other situations where at taller or shorter plant phenotype would be advantageous are readily apparent to persons skilled in the art. Some further examples of applications for this invention are the creation of miniature vegetables and fruit trees; leafy vegetables, tomatoes, peppers etc., and reduction in the flowering time of ornaments (such as orchids).
There are instances where enlargement of a particular part of the plant would be desirable. A particularly good example of this is the advantage conferred to a plant by an expanded root and root hair system. Roots serve not only to anchor the plant in the soil, but also as the means by which the plant absorbs nutrients and water from the soil. This absorption is achieved by the root hairs, fine cellular elongations at the tips of roots. A well-developed root hair system is crucial to plant survival. A plant that has an expanded root system, and in particular an expanded system of root hairs, is better able to absorb nutrients and water from the soil. The enhanced ability to take up water is particularly advantageous for plants grown in drought-prone or water-poor environments, and provides increased tolerance to water stress.
For many crops where the harvest consists of the flower, fruit or seed, yield is effected by the size of the plant at the time of flowering. The larger the plant, the greater is its capacity to flower prolifically and bear a large crop. Thus, delaying the onset of flowering can frequently lead to higher yields by allowing the plant to grow larger before diverting its metabolic energy to flower, fruit and seed production. Conversely, a rapid onset of flowering can be advantageous in an environment where the growing season is relatively short. Early flowering permits a crop to mature for harvest before climatic conditions become unfavorable to plant growth.
According to the present invention, it has been found that over-production of profilin in a plant cell leads to increased cell elongation. At the gross level, the overall effect on the plant is a xe2x80x9ctallxe2x80x9d growth habit and general enlargement of plant parts, including leaves and the root and root hair systems. Over-production of profilin can be achieved in several ways. A preferred way of achieving over-production is to transform a plant or plant cell with a gene construct that expresses high levels of profilin in vivo. High levels of gene expression can be obtained, for example, by using a naturally-occurring highly-expressed profilin coding sequence. Some examples of highly-expressed profilins are Profilin 1 and 2. Another method of achieving high levels of expression is to link a profilin coding sequence to a highly active plant promoter, preferably along with one or more promoter enhancer sequences. Several highly active plant promoters are known in the art, such as the cauliflower mosaic virus (CaMV) 35S promoter, the opine synthase promoters (mannopine synthase promoter, nopaline synthase promoter, etc.) Selective expression of profilin in particular plant tissues can be achieved by the use of tissue-specific promoters known in the art, such as perfect palindronic sequence tetramer fused 10-90 S35 promoter (Salinas et al., 1992). Selection of appropriate promoters and enhancers for use in the invention is within the ordinary skill in the art. Preferably, a combination of the above approaches to enhancing expression is used, e.g. a naturally highly-expressed sequence is linked to an active promoter with one or more enhancer sequences.
Once the gene is obtained, it can be transformed into the host by any conventional means. Examples of plant transformation techniques currently known in the art are the calcium phosphate method, electroporation, microparticle bombardment, Agrobacterium tumefaciens infection, PEG transformation, vacuum infiltration and in planta approach. The selection and use of an appropriate transformation method is well within the skill of the person of ordinary skill in the art. Preferably, stable transformation is achieved, meaning that the introduced sequences are stably integrated into the plant genome (be it into the nuclear, chloroplast or mitochondrial chromosomes, or into two or all of these). A transgenic plant can be grown from a transformed plant cell or cell culture by any of several standard tissue culture and regeneration techniques within the skill of the art. If a growing plant or plant part is transformed, a whole transformed plant can be obtained by methods such as vegetative propagation of the plant part, again by methods within the ordinary skill in the art.
The present invention has also shown that under-production of profilin in a plant cell leads to a decrease in cell elongation and plant part size. The overall effect of this is a plant with a xe2x80x9cdwarfxe2x80x9d growth habit. Like profilin over-production, under-expression of profilins can be achieved in several ways. Preferably, a gene encoding an antisense RNA complementary to the native plant profilin gene is introduced into the plant. A simple way of doing this is to link a plant promoter to a copy of the plant profilin coding sequence oriented in the antisense direction. The xe2x80x9cbackwardsxe2x80x9d coding sequence will be transcribed into an mRNA complementary to the native plant profilin mRNA transcribed from the native coding sequence. The antisense mRNA prevents expression of profilin, most likely by co-suppression of the profilin gene. The techniques of gene construction, transformation and regeneration described above can be equally applied in this embodiment of the invention.
Plant Materials and Growth Conditions
Arabidopsis thaliana C24 ecotype was used. Seeds were surface sterilized with 10% bleach and washed three times with sterile deionized water. After the final wash, 0.2% agarose was added to the seeds and plated on MS media with 3% Sucrose. Plates were incubated at 4xc2x0 C. for two days and then treated with white light for two hours to induce germination. For growth in the dark plates were wrapped with three layers of aluminum foil and kept vertically in a tissue culture room at 22xc2x0. For the cold treatment, the wrapped plants were kept vertically in a refrigerator for 4 weeks. For light growth, plates were exposed to 16 hr. light/8 hr. Dark at 22xc2x0. After three weeks the seedlings were potted in soil and grown in a growth chamber with a photoperiod of 16 hours and humidity of 75%.
Vector Constructions and Plant Transformations
The coding region of profilin-1cDNA (Christensen et al., 1996) was cloned downstream of a 35S promoter contained in a binary plasmid pVIP40 in sense and in antisense orientations. For the profilin-1 promoter-Gus fusion constructs 1.0 Kb of the profilin-1 promoter was cloned in pVIP 40 Vector. Arabidopsis thaliana C24 roots were used as the plant material for transformation. T3 transgenic lines were generated and used to obtain homozygous T4 seeds. Two independent lines for profilin-1 over-expression and three lines for under-expression were used.
Northern Analysis
Total RNA was extracted using a Qigen RNA isolation Kit, from 10-day old dark grown or light grown seedlings of wild type (WT) and transgenic plants. 10 xcexcg of total RNA was used for the WT and the PFN-0 (profilin-over expressing lines) lines and 30 xcexcg for the PFN-U (profilin-under expressing lines) lines were loaded on 1.2% agarose gels and electrophoresed. RNA samples were blotted on to a nylon membrane which was used for hybridizations. Radiolabelled antisense strand of profilin-1 was transcribed from the profilin-1 cDNA cloned in pBlueScript and used as a probe for the hybridizations. Arabidopsis actin-7 cDNA and ADF-1 cDNA were used to make radiolabelled probes for the blots to analyze actin and ADF expression levels in profilin transgenic plants. 18S rRNA was used as an internal control to normalize the loading amounts of RNA samples.
Generation of Antibody, SDS-PAGE and Western Blotting
Profilin-1 and Profilin-3 proteins were expressed as a recombinant protein in E. Coli (Christensen et al., 1996) and was used to immunize the rabbits to obtain polyclonal antibodies. The antisera was affinity purified on a Protein A Sepharose CL-4B (Pharmacia) column and was used for the analysis. Ten day old (light or dark grown) seedlings grown at 22xc2x0 C. or 4 weeks old etiolated seedlings grown at 4xc2x0 C. were used as the plant material for protein extraction for western blots. The plant materials were extracted on ice with 50 mM Tris-Cl (pH 8.0), 0.5 mM Calcium Chloride, 0.5% NP40, 0.5 mM xcex2ME and Aprotinin and Leupeptin (1 xcexcg/ml). The contents were centrifuged for 10 minutes at 4xc2x0 C. and the supernatant was collected. The protein concentrations were determined by Bradford""s method and 20 xcexcg was loaded on 15% polyacrylamide gels (Laemmli, 1970).
Proteins were then transferred to the nitrocellulose membrane and processed further. Membranes were blocked with 3% non-fat milk in PBS buffer containing 0.05% Tween-20 for an hour and washed several times followed by overnight incubation with affinity purified anti-profilin-1. The membranes were then washed with PBS and treated with alkaline phosphatase conjugated anti-rabit IgG (Promega) and incubated for an hour. After several washes the bands were developed using the substrate cocktail. For actin protein and ADF protein level detection we used the antibodies raised against actin. Actin-7 fusion protein was expressed in E. coli and the purified recombinant protein was used to raise antibodies in rabbits and ADF. ADF-1 fusion protein was expressed in E. coli and the purified recombinant protein was used to raise antibodies in rabbits.
Cell Length and Diameter Measurements
Longitudinal and cross sections of wild type and transgenic,seedlings grown for 10 days in the dark were photographed under the light microscope. The length and diameter of the epidermal and cortex cells were measured on the enlarged photograph. More than 50 cells of each plant were used for the measurements.
Flowering Time (FT) and Leaf Number (LN)
FT of wild type and transgenic plants were scored as the number of days from the time when the plates were placed in the tissue culture room to the time of opening of the first flower. Leaf number was scored as the number of leaves on the rosette (excluding cotyledon) and the main flowering stalk at the time of opening of the first flower. Three weeks after germination about 15 randomly selected plants from the wild type and each of the two independent lines of over- and three of the under-expressing transgenic plants were transferred in soil.
Microscopy
Ten day old etiolated seedings in fixation buffer were kept on ice, subjected to 30 sec microwave oven treatment, followed by fixation in 0.1M cacodylate buffer (pH 7.4) containing 4% paraformaldehyde, 5% glutaraldehyde and 0.1M CaCl2 for three hours. The seedlings were then washed with 0.1M cacodylate buffer for 1.5 hours and post-fixed for three hours with 2% osmium tetroxide. Dehydration with an ethanol series and propylene oxide was carried out and embedded in Spurr resin (Electron Microscopy Sciences, Fort Washington, Pa.). Semi-thin sections were stained with toluidine blue for light microscopy analysis.
For scanning electron microscopic observation, the specimens were fixed in PBS buffer (pH 7.3) containing 2.5% glutaraldehyde and 2% paraformaldehyde at 4xc2x0 C. for 2.5 hours, followed by dehydration through a gradient concentration of ethanol. Samples were then dried in liquid carbon dioxide on Samdri-780u (Tousimis Research Corporation, Rockville, Md.), mounted on stubs, sputter coated with gold, and examined with a scanning electron microscope (model JSM-T220A; JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 20 Kv.
xcex2-Glucuronidase (GUS) Assay
Gus assay was performed histochemically with 5 bromo-4-chloro-3-indoyl glucuronide (X-Gluc, Jefferson, 1987) as the substrate using a protocol adapted from Toriyama et al. (1991). The entire seedlings were submerged in the GUS-stain solution (2 mM X-Gluc, 0.1M sodium phosphate buffer, pH 7.0, 0.5% Triton X-100, 2 mM K3[Fe(CN)6], 2 mM K4[Fe(CN)6], and 0.2% NaN3) and vacuum infiltrated for about 10 minutes and then incubated at 37xc2x0 C. overnight. The reaction was stopped and then the plant material cleared by rinsing and incubation with 70% ethanol overnight at 37xc2x0 C.
Actin Staining
Wild type and transgenic etiolated seedlings (10 days old) were placed in an Eppendorf tube containing a slurry of EZE-LAP, lapping compound #700 WF (Scour Pads, Australia), in phosphate buffer (pH 7.2) and vortexed for 1 minute. After washing three times with phosphate buffer, the top portion of seedlings including cotyledons, petioles, hook and a portion of hypocotyl were cut off and incubated in fluorescein-phalloidin (Molecular Probes, U.S.A.) in phosphate buffer for two hours. The specimens were mounted on a glass slide with the same stain solution and cortical actin patterns of epidermal cells were visualized using a confocal microscope (MRC 600 or MRC 1024, BioRad). In case of fixation, the seedlings were incubated in 4% paraformaldehyde in phosphate buffer for 45 minutes and then washed two times with phosphate buffer followed by EZE-LAP treatment as described above.